Forkhead transcription factor Cs-FoxO gene of coccinella septempunctata as well as inhibitor and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a coccinella septempunctata forkhead transcription factor Cs-FoxO gene as well as an inhibitor and application thereof. The invention provides a nucleotide sequence of a coccinella septempunctata forked head transcription factor Cs-FoxO gene as shown in SEQ ID NO. 1 and an amino acid sequence as shown in SEQ ID NO. 2; the invention also provides an application of the forkhead transcription factor Cs-FoxO gene inhibitor in relieving biological diapause. After dsRNA expression inhibition, the forked head transcription factor Cs-FoxO gene provided by the invention can relieve the diapause of the coccinella septempunctata, and the dsRNA of the Cs-FoxO gene can be used as a related preparation and product for relieving the reproductive diapause of the coccinella septempunctata, so that the development of the natural enemy industry is promoted, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to the Cs-FoxO gene of the forkhead transcription factor of the seven-spotted ladybug, its inhibitor and application. Background Art

[0002] The seven-spotted ladybug (Coccinella septempunctata) is an excellent natural enemy insect that can prey on agricultural and forestry pests such as aphids, whiteflies, and psyllids, and is widely used in the biological control of pests. Diapause is a state in which insects enter a state of growth and development arrest in response to changes in the external environment. Diapause brings challenges and opportunities to the application of natural enemy insects in biological control. On the one hand, natural enemy insects can be induced to enter the diapause state to extend the shelf life of natural enemy products; on the other hand, the diapause of natural enemy products can be lifted to meet the control needs of natural enemy insects for pests in a timely manner. Therefore, carrying out research on the diapause regulation technology of the seven-spotted ladybug is crucial for promoting the application of biological control of the seven-spotted ladybug and the development of the natural enemy industry.

[0003] RNA interference (RNAi) is an important gene silencing method discovered in recent years. This technology efficiently and specifically degrades the mRNA of the corresponding sequence through double-stranded RNA (dsRNA). Since the RNAi technology can specifically inhibit the expression of specific genes, this technology has been widely used in the fields of exploring gene functions and gene therapy. This technology uses the insect's own gene fragments to inhibit the expression of key genes in the growth and development of insects through RNAi, thereby controlling the growth, development, and reproduction of insects. The key problem in achieving effective control of insects based on RNAi is to screen target genes that are specific to the target insect and have a significant inhibitory effect on its growth and development. However, the technical difficulty lies in selecting which gene to use as the target for regulation. Summary of the Invention

[0004] The purpose of the present invention is to provide a Cs-FoxO gene of the forkhead transcription factor of the seven-spotted ladybug, and further provide its encoded protein and an inhibitor of the Cs-FoxO gene of the forkhead transcription factor. This inhibitor of the Cs-FoxO gene of the forkhead transcription factor can be used to lift biological diapause.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a Cs-FoxO gene of the forkhead transcription factor of the seven-spotted ladybug, having any one of the following nucleotide sequences:

[0007] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0008] (2)A coding nucleotide sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.1.

[0009]

[0010] Another object of the present invention is to provide a protein encoding the above-mentioned Coccinella septempunctata Forkhead transcription factor Cs-FoxO gene, which has an amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 95% homology with the amino acid sequence shown in SEQ ID NO.2. It should be understood that those skilled in the art can substitute, delete and / or add one or several amino acids to the amino acid sequence disclosed by the present invention without affecting its activity, so as to obtain a mutant sequence of the protein. Therefore, the Coccinella septempunctata Forkhead transcription factor Cs-FoxO protein of the present invention also includes a protein derived from the Coccinella septempunctata Forkhead transcription factor Cs-FoxO protein with the same activity as the Coccinella septempunctata Forkhead transcription factor Cs-FoxO protein, which is obtained by substituting, replacing and / or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.2.

[0011] MLISRNHDYLETSKMSIPCRLQPDHNPPSNSREMRNKAEKLRRDKLNSYIGELAKMVPMVAKSSKRMDKTSILRLSASHLRIYHTLMNGKVKLQIQMPHQVDQCMLEQIVYNELGGFLMVLDANAKIMFVSPTVENLLGYLQTDLMGQSIYNVTLQMDHEILRRHLIISNSTDENCNRDNFMISLKRAAPRSEMPVYERVRVMSVFKSLSYTTDYDTATLMEVPPNTVGNDIWLLFIRMNRPEKIPLRMMESSKDEYYTRHLVDGRIVCCDQRISLIAGYFTDEVFGISAFKFMHLDDVRWVIIALRQMYDRGETKGTSCYRLLSRNSKYIYLRTSGFLEYDSHGTVESFLCYNSLVDETEGKRLIEEMKRRYSAYVNTNYLIPGSPNTPIGAESLEEPLNVEQAIKHLIMNLPSSPCSTPSPKLTYTDEKEDCDEKLTEISTDNKVIPKTVLKRPPCTNLDANLPIKRLKNSPYSMHNPPLTFKTRTDLRIKEEPLCEDLYREI(SEQ ID NO.2)

[0012] Another object of the present invention is to provide an inhibitor of the forkhead transcription factor Cs-FoxO gene, which is used to inhibit the expression of the forkhead transcription factor Cs-FoxO gene, or reduce the level of the forkhead transcription factor Cs-FoxO protein, or degrade the forkhead transcription factor Cs-FoxO protein, or inhibit the dsRNA, miRNA, siRNA, ribozyme or shRNA of the forkhead transcription factor Cs-FoxO gene.

[0013] The dsRNA includes the sequence shown in SEQ ID NO.3 (provided in the form of the sense strand).

[0014] ACGCTTTCAACAACTGGACGGTGCCTGGCATGAGTATGAACATGGTGAAAATGGAGCCTCTGCAGGAGTTGGACGGTTTCGAGCCGCAGACAAGGGCTAGATCCAACACGTGGCCCTTGCCACGGCCTGAAAATTACGTAGAGCCTGGAGATGAAGGAAGCAACAAGTGCGCTGGATTGCCAGGGCCGGCTCAGGCTGCCACACAGGTGCCGGCTAAGAAAAATTCCAGCAGGAGAAATGCCTGGGGTAATCTTAGCTATGCAGACCTGATAACGCAGGCCATCACTTCTT(SEQID NO.3)

[0015] Preferably, the dsRNA is a double-stranded RNA composed of the nucleotide sequence of the forward primer T7-dsCs-FoxO-F (SEQ ID NO.4) and the nucleotide sequence of the reverse primer T7-dsCs-FoxO-R (SEQ ID NO.5).

[0016] The present invention provides a method for preparing an inhibitor of the Cs-FoxO gene, which includes the following steps: using the ORF sequence of the Cs-FoxO gene as a template, amplifying with the forward primer T7-dsCs-FoxO-F and the reverse primer T7-dsCs-FoxO-R, and then synthesizing dsRNA by in vitro transcription. The sequence of the forward primer T7-dsCs-FoxO-F is as shown in SEQ ID NO.4, and the sequence of the reverse primer T7-dsCs-FoxO-R is as shown in SEQ ID NO.5.

[0017] Another object of the present invention is to provide the application of the above-mentioned Cs-FoxO gene inhibitor in relieving biological diapause. By introducing the Cs-FoxO gene inhibitor into an organism, the purpose of relieving biological diapause can be achieved.

[0018] The present invention provides a method for relieving biological diapause, comprising the following steps: introducing the Cs-FoxO gene inhibitor into an organism. The organism may be a seven-spotted ladybug.

[0019] The seven-spotted ladybird forkhead transcription factor Cs-FoxO gene of the present invention is proved by RNAi interference experiment that after the dsRNA sequence of the Cs-FoxO gene is injected into the abdominal intersegmental membrane of the seven-spotted ladybird after emergence, the abundance of Cs-VgmRNA in the seven-spotted ladybird is significantly upregulated, and obvious yolk deposition is found in the egg tubules, indicating that knocking down the Cs-FoxO gene significantly promotes the ovarian development of the diapause female insect, and the dsRNA thereof can be used as a related preparation and product for relieving the diapause of the seven-spotted ladybird, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 qRT-PCR analysis of the expression level of Cs-FoxO gene in Coccinella septempunctata under diapause and non-diapause conditions in newly emerged females (NE), diapause females (D10) and non-diapause females (N10).

[0021] Figure 2 Figure 3 is the effect of RNA interference of Cs-FoxO gene on diapausing female insects, where A is the effect of RNA interference of Cs-FoxO gene on ovarian development of diapausing female insects, B is the relative expression level of CsVg gene in diapausing female insects after RNA interference of Cs-FoxO gene, C and D are the changes of ovarian width and ovarian length after RNA interference of Cs-FoxO gene, respectively, and E is the RNA interference efficiency of Cs-FoxO. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0023] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below in conjunction with the drawings of the specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific technology or conditions are not indicated in the embodiments, they are all conventional methods or carried out according to the technology or conditions described in the literature of the field, or according to the product manual. The reagents and instruments used, etc., which do not indicate the manufacturer, are all conventional products that can be purchased through regular channels.

[0024] In the examples, 2×TransFast Taq PCR SuperMix(+dye)(TransGen Biotech) was used to clone the Cs-FoxO gene, and UPM (purchased from Nanjing Novoprotein Scientific Co., Ltd.) was used for 5′RACE and 3′RACE amplification.

[0025] In the examples, the sequencing primers for the diapause termination gene, the primers for making dsRNA of the diapause termination gene, and the RT-qPCR primers for the diapause termination gene are shown in Table 1. The primers and sequencing were both synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0026] Table 1 Primer sequences and their functions

[0027]

[0028]

[0029] The following is an introduction through specific examples.

[0030] Example 1 Obtaining the ORF sequence of the forkhead transcription factor (Cs-FoxO) gene of Coccinella septempunctata

[0031] Trizol reagent (Invitrogen) was used to extract the total RNA of Coccinella septempunctata. According to the partial sequence (Contig13394) of the Cs-FoxO gene of Coccinella septempunctata, the middle fragment cloning primers Cs-FoxO-F and Cs-FoxO-R, the 3'-RACE primer (Cs-FoxO-3GSP), and the 5'-RACE primer (Cs-FoxO-5GSP) were designed using the Primer 3.0 NCBI online software.

[0032] The SMARTer RACE5' / 3'Kit kit (Takara Biotechnology (Beijing) Co., Ltd.) was used for the synthesis of the first-strand cDNA and subsequent related experiments.

[0033] ① Prepare the 5'-RACE-Ready cDNA and 3'-RACE-Ready cDNA synthesis reaction buffer mixture for the Cs-FoxO gene of Coccinella septempunctata, including: 4.0 μL of 5×First-strand Buffer, 0.5 μL of DTT (100 mM), and 1.0 μL of dNTPs (20 mM).

[0034] ②CsFoxO-F 3'-RACE (total volume 12.0 μL) includes: RNA 1.0 - 11 μL, 3′-CDS Primer A 1.0 μL, Sterile H2O 0.0 - 10.0 μL. After briefly mixing and centrifuging CsFoxO-F 3'-RACE respectively, incubate it in a metal bath at 72 °C for 3 minutes, cool it at 42 °C for 2 minutes, and centrifuge at 14000×g for 10 - 20 seconds.

[0035] ③CsFoxO-F 5'-RACE (total volume 11.0 μL) includes: RNA 1.0 - 10 μL, 5′-CDS Primer A 1.0 μL, Sterile H2O 0.0 - 9.0 μL. After briefly mixing and centrifuging CsFoxO-F 5'-RACE, incubate it in a metal bath at 72 °C for 3 minutes, cool it at 42 °C for 2 minutes, and centrifuge at 14000×g for 10 - 20 seconds. Add 1 μL of SMARTerII A Oligonucleotide to the CsFoxO-F 5'-RACE reaction system, mix well and centrifuge briefly.

[0036] ④Prepare the reaction systems for synthesizing Coccinella septempunctata Cs-FoxO 5'-RACE-Ready cDNA and 3'-RACE-Ready cDNA (both with a total volume of 8.0 μL): Buffer Mix (i.e., reaction buffer mixture) 5.5 μL, RNase Inhibitor (40 U / μL) 0.5 μL, SMARTScribe Reverse Transcriptase (100 U) 2.0 μL. After preparation, add the 8 μL 3'-RACE-Ready cDNA synthesis reaction system to the mixture prepared in step ②, and add the 8 μL Coccinella septempunctata Cs-FoxO 5'-RACE-Ready cDNA synthesis reaction system to the reaction system prepared in step ③. Place it in a PCR instrument, incubate at 42 °C for 90 minutes, and heat at 70 °C for 10 minutes. Obtain the 5'-RACE-Ready cDNA and 3'-RACE-Ready cDNA of the Coccinella septempunctata Cs-FoxO gene.

[0037] Perform RACE experiments successively with 3'-RACE primers (Cs-FoxO-3GSP1, Cs-FoxO-3GSP2, Cs-FoxO-3GSP3) and 5'-RACE primers (Cs-FoxO-5GSP1, Cs-FoxO-5GSP2) (as shown in Table 1) to obtain the complete ORF reading frame sequence of Cs-FoxO.

[0038] Prepare the Master Mix, including: 15.5 μL of PCR-Grade H2O, 25.0 μL of 2× SeqAmp Buffer, and 1.0 μL of SeqAmp DNA Polymerase.

[0039] Prepare the 5'-RACE PCR product, with each specific primer prepared in a separate tube. The 5'-RACE PCR reaction system includes: 2.5 μL of 5'-RACE Ready cDNA, 5.0 μL of 10× UPM, 1.0 μL of Cs-FoxO-5GSP (10 μM) (Cs-FoxO-5GSP1 / Cs-FoxO-5GSP2 are prepared in separate tubes respectively), and 41.5 μL of Master Mix. The 5'-RACE PCR reaction program includes: 94°C for 3 min, 5 cycles; 94°C for 0.5 min, 68°C for 0.5 min, 72°C for 1 min, 20 cycles (the temperature decreases by 0.5°C for each cycle); 94°C for 0.5 min, 58°C for 0.5 min, 72°C for 1 min, 15 cycles; 72°C for 10 min.

[0040] Prepare the 3'-RACE PCR product, with each specific primer prepared in a separate tube. The 3'-RACE PCR reaction system includes: 2.5 μL of 3'-RACE Ready cDNA, 5.0 μL of 10× UPM, 1.0 μL of Cs-FoxO-3GSP (10 μM) (Cs-FoxO-3GSP1 / Cs-FoxO-3GSP2 / Cs-FoxO-3GSP3 are prepared in separate tubes respectively), and 41.5 μL of Master Mix. The 3'-RACE PCR reaction program includes: 94°C for 3 min, 5 cycles; 94°C for 0.5 min, 68°C for 0.5 min, 72°C for 1 min, 20 cycles (the temperature decreases by 0.5°C for each cycle); 94°C for 0.5 min, 58°C for 0.5 min, 72°C for 1 min, 15 cycles; 72°C for 10 min.

[0041] Perform gel extraction and purification on the obtained 3'-RACE PCR product and 5'-RACE PCR product respectively, and send them to the company for sequencing. According to the sequencing results, compare and analyze to obtain the nucleotide sequence encoding the Cs-FoxO gene as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Cs-FoxO gene as shown in SEQ ID NO.2.

[0042] Example 2

[0043] Analyze the expression levels of the forkhead transcription factor Cs-FoxO gene in the ladybug Coccinella septempunctata under diapause and non-diapause conditions. The experimental method includes the following steps:

[0044] Newly emerged female adults were respectively placed under diapause and non-diapause conditions for induction.

[0045] The diapause conditions include: (18±1)°C, (70±10)% RH, and a photoperiod of L:D = 10h:14h.

[0046] The non-diapause conditions include: (24±1)°C, (70±10)% RH, and a photoperiod of L:D = 16h:8h.

[0047] Newly emerged female adults (NE), female adults induced under non-diapause conditions for 10 days (N10), and female adults induced under diapause conditions for 10 days (D10) were respectively taken. Total RNA of the whole insects was extracted using TRIzol reagent from Invitrogen and reverse transcribed. Using the Trans One-Step gDNA Removal and cDNA Kit (TransGen Biotech Co., Ltd., Beijing, catalog number AT311-03), cDNA was reverse transcribed. Using the reverse transcribed cDNA as a template, the relative expression levels of the forkhead transcription factor Cs-FoxO gene in the ladybug Coccinella septempunctata under different conditions and developmental stages were determined by quantitative real-time RT-PCR. The real-time fluorescence quantitative PCR experiment was carried out on a Lightcycler 96 (Roche, CH) instrument, and the fluorescence signal was detected using 5G qPCR Premix.

[0048] The reaction system includes: 1 μL of template, 0.8 μL of forward primer (10 μM), 0.8 μL of reverse primer (10 μM), 1 μL of 5G qPCR Premix, and 1 μL of RNase free water.

[0049] The reaction parameters include: 95°C for 5 min for hot start; 98°C for 3 s, 58°C for 5 s, for 40 cycles.

[0050] Each reaction was set with 3 technical replicates. The RT-qPCR data was analyzed using the 2 -ΔΔCt -method, ΔΔCt = (Ct 目标基因 -Ct 内参基因 ) 实验组 -(Ct 目标基因 -Ct 内参基因 ) 对照组, where the Actin of Coccinella septempunctata was used as the internal reference gene, and the GraphPad Prism 9.0 software was used to standardize the gene expression and plot the graph. The transcriptional level of Cs-FoxO in NE was used as the standard for normalization to analyze the Cs-FoxO mRNA abundance in N10 and D10.

[0051] The experimental results showed that: compared with N10, the expression level of Cs-FoxO mRNA in D10 was significantly up-regulated by 2.38-fold ( Figure 1 ).

[0052] Example 3 Obtaining dsRNA of the forkhead transcription factor Cs-FoxO gene of Coccinella septempunctata

[0053] Based on the ORF sequence of the forkhead transcription factor (Cs-FoxO) of Coccinella septempunctata obtained in Example 1 (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Cs-FoxO gene is shown in SEQ ID NO.2), dsRNA primers were designed using Primer premier 6. software.

[0054] The target fragment of Cs-FoxO was amplified. The forward primer was T7-dsCs-FoxO-F, the reverse primer was T7-dsCs-FoxO-R, and the template was the cDNA obtained by extracting total RNA and reverse transcription in Example 1. The reaction system (total volume 50 μL) included: template cDNA 2 μL, T7-dsCs-FoxO-F (10 μM) 2 μL, T7-dsCs-FoxO-R (10 μM) 2 μL, 2×TransFast Taq PCR SuperMix (+dye) (TransGen Biotech) 25 μL, RNase free water 19 μL. The reaction conditions included: 94 °C, 3 min; 94 °C, 10 s, 55 - 60 °C 20 s, 72 °C, 30 s, 35 cycles; 72 °C, 5 min.

[0055] Amplify the target fragment of GFP (the sequence is shown in SEQ ID NO. 19). The forward primer is T7-dsCs-GFP-F, and the reverse primer is T7-dsCs-GFP-R. The template is the cDNA of the strain containing GFP stored in the laboratory where the inventor is located (which can be obtained by the public and is only used for non-commercial purposes to repeat the examples described in the present invention). The reaction system (total volume 50 μL) includes: 2 μL of template, 2 μL of T7-dsCs-GFP-F (10 μM), 2 μL of T7-dsCs-GFP-R (10 μM), 25 μL of 2×TransFast Taq PCR SuperMix (+dye) (TransGen Biotech, Beijing), and 19 μL of RNase free water. The reaction conditions include: 94 °C for 3 min; 94 °C for 10 s, 55 - 60 °C for 20 s, 72 °C for 30 s, for 35 cycles; 72 °C for 5 min.

[0056] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG(SEQ ID NO.19)

[0057] Purify the amplified PCR product, and use the T7 RiboMAX TM Express RNAi System (Promega) kit to transcribe DNA in vitro to synthesize dsRNA, named dsCs-FoxO (as shown in SEQ ID NO.3). Use green fluorescent protein GFP as a negative control to synthesize dsGFP. After measuring the concentration of the synthesized dsRNA using NanoDrop2000 (Invitrogen), dilute it with DEPC water to a final concentration of 2 μg / μL and store it in an -80 °C refrigerator for later use.

[0058] Example 4 Effect of RNA interference of the forkhead transcription factor Cs-FoxO gene of the seven-spotted ladybug on diapausing females

[0059] Female seven-spotted ladybugs with consistent developmental progress (within 48 h after eclosion) under non-diapause conditions (i.e., normal conditions) were selected for microinjection. The non-diapause conditions included: (24 ± 1) °C, (70 ± 10)% RH, and a photoperiod of L:D = 16 h:8 h).

[0060] Inject 2 μg of dsCs-FoxO double-stranded (prepared by the method of Example 3) into the abdominal intersegmental membrane of the seven-spotted ladybug, and use female insects injected with an equal amount of dsGFP as a control. The injected seven-spotted ladybugs were reared under diapause conditions (photoperiod L:D = 10 h:14 h; humidity 70%; temperature 18 °C). After 72 hours of interference treatment, total RNA of the seven-spotted ladybug was extracted using the TransZol Up Plus RNA Kit (TransGen Biotech Co., Ltd., Beijing), and the extracted RNA was then used for reverse transcription to synthesize cDNA. Real-time fluorescence quantitative PCR experiments were performed on a Lightcycler 96 (Roche, CH) instrument, and fluorescence signals were detected using 5G qPCR Premix.

[0061] The reaction system included: 1 μL of template (cDNA), 0.8 μL of forward primer (10 μM), 0.8 μL of reverse primer (10 μM), 1 μL of 5G qPCR Premix, and 1 μL of RNase free water.

[0062] The reaction parameters included: 95 °C for 5 min for hot start; 98 °C for 3 s, 58 °C for 5 s, for 40 cycles.

[0063] Each reaction was set with 3 technical replicates. RT-qPCR data was analyzed using the 2 -ΔΔCt method, ΔΔCt = (Ct 目标基因 − Ct 内参基因 ) 实验组 − (Ct 目标基因 − Ct 内参基因 ) 对照组 , where Actin of the seven-spotted ladybug was used as an internal reference gene, and the expression level of the control group injected with dsGFP was used for normalization. The gene expression was normalized and plotted using GraphPad Prism 9.0 software.

[0064] The results of real-time fluorescence quantitative PCR showed that the CsFoxO mRNA level in female insects injected with dsRNA of Cs-FoxO decreased by 89.6% on the 3rd day after injection ( Figure 2 E).

[0065] After injecting dsCs-FoxO double-stranded DNA by the same method as above, the treated female insects were placed under diapause induction conditions for 72 h. The diapause induction conditions included: (18±1)°C, (70±10)% RH, and a photoperiod of L:D = 10 h:14 h. Then they were taken out, RNA was extracted and reverse transcribed into cDNA for fluorescence quantification.

[0066] The research results showed that knocking down the Cs-FoxO gene significantly promoted the ovarian development of diapausing female insects, and there were obvious signs of yolk deposition in the ovarioles ( Figure 2 A), a significant up-regulation of the CsVg mRNA abundance in their bodies ( Figure 2 B), and a significant increase in both the length and width of the ovaries ( Figure 2 C, Figure 2 D), indicating that the dsRNA of the Cs-FoxO gene plays an important role in the termination of diapause in Coccinella septempunctata.

[0067] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. The Cs-FoxO gene of the forkhead transcription factor of Coccinella septempunctata, characterized in that, Having any one of the following nucleotide sequences: (1) The nucleotide sequence shown in SEQ ID NO.1; (2) A coding nucleotide sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.

1.

2. A protein encoding the Cs-FoxO gene of the ladybug forkhead transcription factor as recited in claim 1.

3. The protein according to claim 2, wherein Having the amino acid sequence shown in SEQ ID NO.2, or having an amino acid sequence with at least 95% homology to the amino acid sequence shown in SEQ ID NO.

2.

4. An inhibitor of the forkhead transcription factor Cs-FoxO gene, characterized in that, Comprising RNA, said RNA having any one or more of the functions (1) to (5); (1) For inhibiting the expression of the gene recited in claim 1; (2) For reducing the level of the gene recited in claim 1; (3) For degrading the gene recited in claim 1; (4) For reducing the level of the protein recited in claim 2 or 3; (5) Degrading the protein recited in claim 2 or 3.

5. The forkhead transcription factor Cs-FoxO gene inhibitor according to claim 4, characterized in that The inhibitor of the Cs-FoxO gene of the forkhead transcription factor is selected from one or more of dsRNA, miRNA, siRNA, ribozyme, shRNA that inhibit the Cs-FoxO gene of the forkhead transcription factor.

6. The forkhead transcription factor Cs-FoxO gene inhibitor according to claim 5, wherein The dsRNA comprises the sequence shown in SEQ ID NO.

3.

7. A preparation method of a forkhead transcription factor Cs-FoxO gene inhibitor, characterized in that, Comprising the following steps: Using the ORF sequence of the Cs-FoxO gene recited in claim 1 as a template, amplifying and then in vitro transcribing to synthesize dsRNA with the forward primer T7-dsCs-FoxO-F and the reverse primer T7-dsCs-FoxO-R. The sequence of the forward primer T7-dsCs-FoxO-F is as shown in SEQ ID NO.4, and the sequence of the reverse primer T7-dsCs-FoxO-R is as shown in SEQ ID NO.

5.

8. Use of the inhibitor of the Cs-FoxO gene of the forkhead transcription factor according to any one of claims 4-6 in relieving biological diapause.

9. The application according to claim 8, wherein Introducing an inhibitor of the Cs-FoxO gene of the forkhead transcription factor into an organism.

10. A method for relieving biological diapause, characterized in that: Comprising the following steps: Introducing the inhibitor of the Cs-FoxO gene of the forkhead transcription factor according to any one of claims 4-6 into an organism.

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